William E. Collins

Structure and expression of the gene for Pv200, a major blood-stage surface antigen of Plasmodium vivax

Molecular cloning and structure analysis of the gene encoding the Pv200 protein of the Sal-1 strain of Plasmodium vivax revealed an overall identity of 34-37% when the deduced amino acid sequence was compared with the sequences of various major merozoite surface antigens of Plasmodium falciparum, Plasmodium yoelii and Plasmodium chabaudi. When the Sal-1 Pv200 sequence was compared with the corresponding sequence from the Belèm strain of P. vivax, it was found that the two merozoite surface antigens were relatively well conserved with an overall amino acid sequence identity of 81%. A region of 23 repeated glutamine residues, found in the sequence of the Belèm isolate was not found, however, in the Sal-1 sequence. Amino- and carboxy-terminal domains of the Pv200 protein were expressed in the yeast Saccharomyces cerevisiae. Each recombinant protein was shown to react with antibodies in sera from splenectomized Bolivian Saimiri monkeys that had been infected previously with P. vivax, and in human sera from individuals with a history of exposure to vivax malaria. The availability of recombinant DNA-derived Pv200 proteins will now allow a full assessment of their utility in the diagnosis and immunoprophylaxis of the benign tertian malaria associated with P. vivax infection.

Sequence variations in the non-repetitive regions of the liver stage-specific antigen-1 (LSA-1) of Plasmodium falciparum from field isolates☆☆☆

Liver-stage-specific antigen-l (LSA-1) of Phsmodium falciparum is a 200-kDa protein localized in the parasitophorous vacuole space of liver-stage parasites [1,2]. It is composed of a large central repetitive region and two flanking short non-repetitive Nand C-terminal regions [3]. This antigen is of considerable interest in vaccine development, because two epitopes (Ls6, Ls8) of ISA-1 have been shown to generate cytotoxic T-cell (CTL) responses in HLA-B53 and HLA-B35 positive individuals 141,

Blood and sporozoite stage-specific small subunit ribosomal RNA-encoding genes of the human malaria parasite Plasmodium vivax

Malaria parasites, unlike other eukaryotes, have developmentally controlled distinct small subunit ribosomal RNA (SSUrRNA)-encoding genes (SSUrDNA), sporozoite stage-specific C and blood stage-specific A genes. This report describes characterization of the C and A forms of SSUrDNA from the human malaria parasite Plasmodium vivax. We have aligned and compared these sequences with the reported SSUrDNA sequences of other human malaria parasites to identify the regions with potential for diagnostic probes. The comparison revealed the presence of seven conserved regions (> or = 90% similarity), four highly variable regions (< 60% similarity) and three semiconserved regions. The analysis also revealed that the A and C genes of P. vivax share more similarity with each other, as compared to the A and C genes of P. falciparum. Comparison of the SSUrDNA of human, monkey and rodent malaria parasites revealed that the A genes share more similarity with each other than the C genes share with each other.

Circumsporozoite protein gene of Plasmodium simium, a Plasmodium vivax-like monkey malaria parasite

The circumsporozoite (CS) protein, a species-specific sporozoite surface protein and a vaccine candidate antigen, has been characterized from malaria parasites that naturally cause infections in humans, monkeys and chimpanzees [1-7]. Because of the related structural features of repetitive and nonrepetitive sequences the CS protein genes have also been used as markers for studies of evolutionary relatedness of malaria parasites [8]. The CS protein genes of the nonhuman primate malaria parasites Plasmodium brazilianurn and Plasmodium reichenowi, which are considered to be evolutionarily related to the human malaria parasites Plasrnodium malariae and Plasmodium falciparum, respectively, have been characterized and shown to bear structural and antigenic similarity [4,7]. Plasmodium simium is a nonhuman malaria parasite both morphologically and biologically similar to the human malaria parasite Plasmo-

Characterization of Plasmodium falciparum cloned lines with respect to gametocyte production in vitro, infectivity to Anopheles mosquitoes, and transmission to Aotus monkeys

The production of gametocytes in vitro and their subsequent infectivity to mosquitoes by 3 cloned lines of Plasmodium falciparum were studied. 2 of the cloned lines, Honduras I-clone B3 and Indochina III-clone W2, produced mature gametocytes (stage V) that were infective to Anopheles mosquitoes. The third clone, Sierra Leone I-clone D6, produced gametocytes, the majority of which did not develop beyond stage III. Fully mature gametocytes of Sierra Leone I-clone D6 were not infective to mosquitoes. Sporozoites collected from An. freeborni infected with Honduras I-clone B3 were used in transmission studies. Two of three Aotus monkeys were infected after prepatent periods of 19 and 20 d, respectively. This study supports previous reports that cloned lines of P. falciparum contain the full genetic capacity to produce morphologically mature gametocytes. The transmission to Aotus monkeys has also conclusively established that biologically competent gametocytes of both sexes are produced by clones.

Plasmodium fragile: Detection of a ring-infected erythrocyte surface antigen (RESA)

A ring-infected erythrocyte surface antigen (RESA) has been detected by modified immunofluorescence assay in erythrocytes infected with the simian malaria parasite, Plasmodium fragile. This RESA, of Mr 95,000, shares many characteristics with the RESA initially found in the human malaria parasite P. falciparum. Both antigens are found in the membrane of erythrocytes infected with young asexual parasite stages, in merozoite-enriched preparations, and in parasite culture supernatant. Since the RESA of P. falciparum has been shown to confer protective immunity and since P. fragile infection of rhesus monkeys mimics P. falciparum infection in humans, the finding of a RESA in P. fragile underlines the importance of this species as an animal model for antimalarial vaccines.

Plasmodium fragile: Inhibition of cultures by serum from Rhesus monkeys immunized with homologous parasites

Rhesus monkeys, Macaca mulatta, that had previously been immunized with the Nilgiri strain of Plasmodium fragile grown in culture, together with control monkeys with and without inoculation of Freunds adjuvant, were challenged with cultured parasites. After treatment with chloroquine, the monkeys were rechallenged. Serum specimens from three immunized monkeys caused a specific, dose-dependent inhibition of parasite growth in culture. Fifty percent inhibition of in vitro growth was obtained using 5% immune serum combined with 10% normal rhesus serum. The specific inhibitory component of immune serum was shown to be IgG antibody. Results of the study demonstrated that there is good correlation between the inhibitory activity of immune serum, parasite growth in vitro, the in vivo response to challenge, and the indirect fluorescent antibody titer.

Plasmodium cynomolgi: Immunization of a rhesus monkey with exoerythrocytic stages cultured in autologous hepatocytes

To investigate the immune response to exoerythrocytic stages of malaria parasites, a rhesus monkey was immunized with autologous primary hepatocyte cultures infected with 7-day-old liver stage parasites of Plasmodium cynomolgi. A primary antibody response against EE stage antigens was obtained, and boosted after injection of homologous viable sporozoites. Antibodies directed against sporozoites and blood stages were also detected. The polyvalent immune response observed demonstrates the antigenicity of the liver stages and suggests their involvement in the general immune response against malaria.